1. Field of the Invention
The present invention relates to an immunologic assay in which an antigen/antibody immune complex is formed and isolated through the use of a biotin/avidin association reaction.
2. Brief Description of the Background Art
Immunologic assays for the detection of antigenic substances or antibodies in specimens are well known. For example, Chu (U.S. Pat. No. 4,289,747) discloses an immunometric assay (also known as a "sandwich assay") for the detection of various substances in body fluids. The assay described in the Chu patent utilizes unrestricted polyclonal antisera from different species to form the sandwich with the particular antigenic substance. The sandwich is then removed from the solution via a lectin/sugar association reaction. The assay emphasizes taking advantage of the low binding affinity between lectin and sugar to allow reversible release of the immune complex from the solid phase to which the lectin or sugar is bound. The assays are described in the simultaneous, reverse, and forward modes.
Wolters, et al., U.S. Pat. No. 4,343,896, also describes a sandwich assay. The sandwich is formed in liquid phase between a particular antigen and antibodies raised to that antigen in two species. Reaction between these two groups of antibodies, one of which is labeled, and the antigen is allowed to occur in liquid phase before being exposed to a solid phase matrix to which is bound a second antibody (anti-globulin) reactive to the unlabeled species of antibody forming the immune complex.
In Parikh, et al., U.S. Pat. No. b. 4,298,685, a non-sandwich type assay is described which utilizes biotin and avidin. .This patent discloses a competition assay wherein antigen which may be present competes with enzyme-labeled antigen for binding sites on biotinylated antibody. After incubation of these reagents, the liquid phase is exposed to a solid phase bound avidin to allow removal of the antibody/antigen complexes from the liquid phase.
Another example of a sandwich immunoassay is described in David, et al., U.S. Pat. No. 4,376,110. The assay disclosed here uses two different monoclonal antibodies from two different hybridomas. The David technique involves one of the monoclonal antibodies being bound to the solid phase throughout the assay, rather than being used in a homogeneous phase. The solid phase monoclonal antibody 1 is reacted with the liquid phase to detect the possible presence of antigen. A monoclonal antibody 2, which is labeled and soluble in liquid phase, will form, in the presence of antigen, a sandwich between solid phase monoclonal antibody 1, antigen, and labeled monoclonal antibody 2.
Katz, et al., U.S. Pat. No. 4,496,654, describe a method which utilizes the biotin-avidin reaction to strongly bind antibody to a solid phase substrate. This patent discloses coating an avidinated solid phase with biotinylated antibody for a specific antigen. This antibody-coated solid phase is then reacted with a sample containing the specific antigen and soluble labeled antibody to the specific antigen. After incubation, the labeled antibody bound to the solid phase or remaining in solution is measured.
In Gallati, et al., United Kingdom Patent Application GB No. 2,074,727A, sandwich assays are described which may occur in liquid phase between (1) two monoclonal antibodies specific for two different epitopes of the same antigen, (2) a monoclonal antibody and polyclonal antibodies raised in another species to the same antigen, and (3) polyclonal antibodies raised in two different species and being directed towards different epitopes of the same antigen.
An assay for the determination of allergen-specific human IgE is disclosed in Bennich, et al., U.S. Pat. No. 3,720,760. This patent described an assay in which a sample is exposed first to a solid phase to which is bound a specific allergen, followed by addition of a radioactively iodinated anti-globulin to human IgE.
The previously described immunoassays fall into two main categories. First, there are those immunoassays (David, Bennich, Katz) wherein one of the binding pair, either an antigen or an antibody, is attached to a solid phase, and a second binding partner, which is labeled, usually an antibody, is in the liquid phase. If the specimen contains the substance to be detected, then a complex is formed composed of the solid phase first binding partner, the substance being detected, and the detectably labeled second binding partner. In the second category (Chu, Wolters, Gallati) are those immunoassays wherein the first and second binding partners both react freely in the liquid phase to form an immune complex if the specimen contains the substance to be detected. This immune complex is then removed from the liquid phase by binding of one of the binding partners to a carrier which is modified such that it will bind the immune complex. This second type of immunoassay wherein initially neither binding partner is bound to a solid phase will be denominated herein as Delayed Solid Phase (DESP).
One of the ma]or, problems with prior art DESP assays is that the systems use non-restricted polyclonal antibodies as the first and second binding partners in liquid phase. As a consequence, these two populations of antibodies will often compete for the same binding sites (epitopes) on the antigen in solution and thus lower the overall sensitivity of the assay. An additional problem with these prior art DESP immunoassays is that the ligand reaction used to remove the immune complex from solution is of low to moderate affinity.
None of the DESP assays described in the prior art disclose a system utilizing a monoclonal antibody as a first binding partner and a detectably labeled second binding partner, which is either a different monoclonal antibody or a polyclonal antibody of restricted specificity, used in conjunction with a high affinity ligand for removal of the immune complex from the liquid phase.